Minoru S. H. Ko,Laboratory of Genetics,National Institute on Aging,NIH,Baltimore,MD 21224,USA, Tel. 410-558-8359. E-mail: kom@mail.nih.gov
Treatment protocol
Day 1: Cells were plated in regular ES medium with Doxycycline (0.2ug/ml) and Puromycin (1.5ug/ml). Approximately 18 hours after plating, the cells were rinsed once with PBS, then the medium was replaced with warmed medium without Doxycycline. Three hours after the first medium change, the medium was changed again. Finally, at three hours after the second medium change, the medium was replaced for a final time. At 24 hours after the final wash, the cells were harvested in Trizol. There was no medium change on the day of RNA harvest.
Extracted molecule
total RNA
Extraction protocol
The samples were dissolved in the well with TRIzol reagent 1/2 mL. They were then transferred to Phase Lock Gel Heavy tubes (eppendorf) and 0.2 volumes of chloroform (Sigma-Aldrich) was added to each sample. They were mixed by hand and allowed to stand for 2-5 min at room temperature. The samples were then separated by centrifugation for 5 minutes at 15,300 rpm on an eppendorf centrifuge at 4C. The aqeous phase was transferred to another eppendorf tube, and 0.8 volumes of ice-cold isopropanol was added. They were allowed to incubate for a minimum of 10 minutes at room temperature. If longer, they were placed at 4C. The sample was centrifuged again at the same conditions for 15 minutes. After centrifugation, the liquid was dumped out of the tubes and the pellet was washed with 1 mL of ice-cold 70% ethanol. The tube was spun for 5 minutes, then the ethanol was dumped out. Excess was removed with a pipette and the pellet was allowed to air dry until no liquid remained, but no longer. ddH2O was then added in the appropriate volume, the sample was allowed to dissolve for 5 minutes at 55C and then transferred to -80C for storage.
Label
Cy3
Label protocol
Total RNA was labeled using Agilents Low RNA Input Fluorescent Linear Amplification kit, according to manufacturers instructions.
Total RNA was extracted from cultured cells using a Stratagene Absolutely RNA kit. DNase-treated total RNA samples from 11 cultured cell lines derived from embryo, embryo fibroblast, kidney, liver/hepatocyte, lung/alveolar macrophage, B-lymphocyte, T-lymphocyte (thymus), mammary gland, muscle myoblast, skin, and testis were pooled in equal parts. The resulting URM RNA was mixed with 129ES cell RNA (Lif+) at 2:1 ratio and 2.5ug of mixed RNA was used for labeling in each tube.
Label
Cy5
Label protocol
Total RNA was labeled using Agilents Low RNA Input Fluorescent Linear Amplification kit, according to manufacturers instructions.
Hybridization protocol
Slides were hybridized according to manufacturers protocol (Two-Color Microarray-Based Genr Expression Analysis Protocol, Product # G4140-90050, Version 5.0.1, August 2006)
Scan protocol
Slides were scanned with an Agilent DNA Microarray Scanner (model G2505-64120) at 100% and 10% PMT in both channels, with a scan resolution of 5um.
Description
T-24h
Data processing
Data are extracted with Agilent Feature Extraction Software.The data were further processed with NIA ANOVA tool utilities.See http://lgsun.grc.nia.nih.gov/ANOVA for details. The data is normalized with the following method: (1) Take values from the columns gDyeNormSignal,rDyeNormSignal in the raw files and log-transform them using: log10(max(x,1)). These values will be referenced below as Xgi and Xri where i is array number. (2) Take average of Xri's for the oligo among 124 arrays in the series: AverXr = average(Xri). (3) For each array estimate Yi = Xgi-Xri+AverXr (4) Round Yi to 4 decimal digits. This is used as the normalized VALUE. More detailed information with error correction can be found at http://lgsun.grc.nia.nih.gov/ANOVA/help.html#arrayjoin
